The present invention relates to particle discrimination by light scattering, and more particularly to a flow cytometer and method therefore that discriminates particles employing a high numerical aperture. Numerical aperture is defined as the refractive index of the medium through which light is collected multiplied by the sine value of one-half of the angle of light collection.
The discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles in a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
One method of the above is disclosed in U.S. Pat. No. 5,017,497 issued to de Grooth et al. Referring to FIG. 1, the ""497 Patent discloses a flow cell 2 through which cells from, for example, blood or the like, flow substantially one by one therethrough. A laser input 4 emits a polarized beam of laser light that is oriented substantially orthogonally to the flow of blood cell through flow cell 2 such that the polarized laser light impinges upon the blood cells as they pass through flow cell 2. By xe2x80x9cpolarizedxe2x80x9d it is meant that the plane of the electric field oscillation of the laser light is uniform. An optical lens 6 has an numerical aperture of 0.6 which limits the cone of scattered light from the blood cells that can be collected to 72xc2x0 or less, and practically to 50xc2x0 as disclosed in the ""497 Patent. The central axis of the cone of lens 6 is 90xc2x0 to both the path of the polarized laser light and the flow of blood cells through flow cell 2. The scattered light emanating from lens 6 is collimated in a manner known in the art. The scattered light now has a mixed polarization that is characteristic of the cell type. The light next passes through a beam splitter 8 that divides the light into two separate beams. A first light beam, substantially concentric with the light beam that originally emanated from lens 6, passes through first polarization analyzer 10. Polarization analyzer 10 is configured to pass therethrough only polarized light having a vector the same as the original laser light. The second beam emanating from beam splitter 8 is oriented substantially perpendicular to the orientation of the first beam emanating from beam splitter 8. This second beam enters second polarization analyzer 12. Second polarization analyzer 12 is configured to pass therethrough only light having a polarization vector substantially orthogonal to the polarization vector of the other beam from beam splitter 8 that passed through first polarization analyzer 10. The beams that pass through first polarization analyzer 10 and second polarization 12 enter polarized detector 14 and depolarized light detector 16, respectively. The ratio of the outputs of polarized light detector 14 and depolarized light detector 16, based on intensity, provide the depolarization ratio.
As shown in FIG. 4 eosinophils, a subset of leukocytes (white blood cells), depolarize the right angle of scattered light quantified by the above configuration to a greater degree than other leukocytes. FIG. 4 is a graphical representation having the output of polarized light detector 14 as one axis and the output of depolarized light detector 16 as the axis. While the above invention does provide some useful data regarding leukocytes, and more specifically eosinophils, as shown in FIGS. 6B, 7B, 8B and 9B, the cluster points within the eosinophil cluster (the cluster points above the angled threshold line on the graphical representation having xe2x80x9cDEPOLxe2x80x9d as one axis and xe2x80x9cORTHAGONALxe2x80x9d as the other axis) are quite condensed. The dense nature of the points within the eosinophil cluster results in difficulty for the computer software programs that ascertain and identify clusters to accurately identify eosinophil clusters. Additionally, this prior art configuration requires expensive optical devices such as photo multiplier tubes, and lens 6, first polarization amplifier 10 and second polarization amplifier 12.
The prior art as indicated in the ""497 Patent is unable to distinguish eosinophils without utilizing polarized and depolarized light methods, because the cone of light collected is 72xc2x0 or less, based on the numerical aperture of the light collection lens, and more practically 50xc2x0, based on the number of optical elements that are used. These optical elements, such as beam splitter 8, polarization analyzers 10 and 12, and light detectors 14 and 16, contribute to reducing the effective light collection of the system is substantially less that 72xc2x0, and to more practically, 50xc2x0.
Copending U.S. patent application Ser. No. 09/507,515 discloses a device and method for distinguishing eosinophils in a sample of blood cells. The device uses a right angle scatter light detector that is effective to collect a cone of unfiltered right angle scattered light of at least 100xc2x0 and convert the collected right angle scattered light into a right angle scattered light signal. This signal is processed by the device to distinguish eosinophils from other leukocytes in the sample on the basis of the right angle scattered light signal.
While the device described in the ""515 application is capable of detecting eosinophils using unfiltered right angle scattered light, its properties create problems for the economical production of the device. Mechanically, providing a solid mounting scheme to keep right angle scatter detector 22 in place at a very small distance to flow cell 18 is difficult to design, and more difficult to manufacture. Also, minimizing the distance from right angle detector 22 to pre-amplifier 26 is essential to eliminate electrical noise that would otherwise be picked up by the leads of the right angle photo detector 22.
A need thus exists for a flow cytometer apparatus and related method in which the cell cluster points are less dense for ease of characterization of the different cell clusters. A need also exists for the above apparatus and method which has fewer and less expensive components, and which is easy and economical to manufacture.
The lens-less light collection flow cytometer of the present invention includes a flow cell and a laser input. The laser input emits a beam of light that is oriented substantially orthogonally to the flow of blood cells through the flow cell such that laser light impinges upon the blood cells as they pass through the flow cell. Unlike the prior art, the laser light emitted by the laser input need not be polarized for analysis of the cells according to the present invention. A portion of the beam from the laser input that impinges upon the blood cells in the flow cell is scattered at a substantially right angle to the beam of laser input (xe2x80x9cright angle scatter lightxe2x80x9d). A second portion of the beam from the laser input that impinges upon the cells in the flow cell is scattered at a much lower angle than 90xc2x0. This scatter is termed xe2x80x9cforward scatter lightxe2x80x9d and is collected on two distinct photo detectors. The first detector represents xe2x80x98forward scatter lowxe2x80x99 (FSL), i.e., forward scatter light which has an angle of from about 1xc2x0 to about 3xc2x0 relative to the laser beam input. A second detector represents xe2x80x98forward scatter highxe2x80x99 (FSH), i.e., forward scatter light which has an angle of from about 9xc2x0 to about 12xc2x0 relative to the laser beam input A third photo detector, axial with the impinging laser light, is placed in between these two forward scatter detectors. This detector measures axial light loss, or light extinction (EXT), which is the sum of all the light that is absorbed and scattered by the blood cells. A right angle scatter light detector is oriented to receive the previously mentioned right angle scatter light. The right angle scatter light detector is preferably located about 2 millimeters from the blood cells in the flow cell. An important aspect of the present invention is that, at the distance of about 2 millimeters from the blood cells, the right angle scatter light detector collects a cone of scattered light of at least 100xc2x0 or greater, and preferably 130xc2x0 or greater. It is this larger light cone value over the prior art light cone of about 50xc2x0 in practice, and no greater then 72xc2x0 in theory, that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 50xc2x0 cone of the prior art results in missed signals and lesser cluster separation.
The maximum 72xc2x0 cone of the prior art is mathematically limited to the numerical aperture of the initial light collection lens 6. The numerical aperture for the prior art total optical system shown in FIG. 1 is much lower, because each optical element reduces the numerical aperture of the total optical system. In contrast, the apparent numerical aperture of a lensless light detection system is equal to the numerical aperture of the total optical system. When a lens less optical system is employed, a xcx9c72xc2x0 cone angle produces cluster separations that are not apparent in the prior art. Thus, a lens less light collection system may be used in a flow cytometer, which has a much lower numerical aperture, but maintains cluster separation of eosinophils. The advantage of this device is that a lower numerical aperture system can be produced more efficiently and more reproducibly than the prior art devices, because there is more working distance to place components so that the photodetector is held at the proper distance from the flow cell. Additionally, this extra working distance allows for the detector to be mounted on a printed circuit board, which reduces electronic noise, improving the overall signal-to-noise ratio of the opto-electrical system.
A forward light detector array is oriented to capture the previously mentioned forward scatter low, forward scatter high, and axial light, from the beam of the laser input.
In one embodiment of the present invention, both a right angle scatter light detector and any of the three forward light detectors are employed in order to produce a 2-dimensional cytogram. However, it should be noted that in another embodiment of the present invention, only a right angle scatter light detector is employed, forward light detector is not employed, and characterization of eosinophils is possible.